Gluconate dehydratase

ABSTRACT

A novel gluconate dehydratase derived from  Achromobacter xylosoxidans  and a gene encoding the gluconate dehydratase are provided. By reacting the gluconate dehydratase or a transformed cell containing the gene with an aldonic acid, the corresponding 2-keto-3-deoxyaldonic acid can be efficiently produced.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel gluconate dehydratase capableof efficiently producing a 2-keto-3-deoxyaldonic acid from aldonic acid,to a base sequence encoding the gluconate dehydratase, to a plasmidcontaining the base sequence, and to a cell transformed by the plasmid.The present invention also relates to a process with use of thegluconate dehydratase or the transformed cell for producing a2-keto-3-deoxyaldonic acid and a 2-deoxyaldonic acid or 2-deoxyaldosewhose carbon number is reduced by 1.

2. Description of the Related Art

2-keto-3-deoxyaldonic acids are useful as pharmaceutical material. Forexample, 2-keto-3-deoxyaldonic acids are decarboxylated to2-deoxyaldonic acids or 2-deoxyaldoses whose carbon number is reducedby 1. These substances are thought of for raw materials of antibiotics,antiviral agents, antisense drugs, and other drugs and medicines.

On the other hand, enzymes have been known which catalyze a reaction forproducing 2-keto-3-deoxyaldonic acids by dehydration of aldonic acids.For example, a gluconate dehydratase (EC4.2.1.39) for dehydratingD-gluconic acid to synthesize 2-keto-3-deoxy-D-gluconic acid is derivedfrom Clostridium pasteurianum (Analytical Biochemistry 61, 275 (1974)),Alcaligenes sp. strain M250 (Methods in Enzymology 41, 99 (1975)), orSulfolobus solfataricus (Biotechnol. Lett. 8,497 (1986)). Unfortunately,it is reported that the gluconate dehydratase derived from Clostridiumpasteurianum is liable to oxidize with air and is thus rapidlydeactivated in the presence of air. The gluconate dehydratase derivedfrom Alcaligenes sp. strain M250 is not thermally stable. A report hastaught that the dehydration activity of the gluconate dehydratase is 50%degraded in storage at 0° C. for 7 days in atris(hydroxymethyl)aminomethane (hereinafter referred to as Tris) buffersolution (pH 8.0 to 8.8) containing 1 mM of sodium D-gluconate and 1 mMof magnesium chloride. Hence, these microorganisms or enzymes derivedfrom these microorganisms are not suitable for industrial productionbecause of the difficulty in handling, such as degradation of thereactivity in a process. The gluconate dehydratase derived fromSulfolobus solfataricus has not yet purified, and accordingly, itsscientific characteristics have not been known. For synthesis of2-keto-3-deoxy-D-gluconic acid with use of the Sulfolobus solfataricusas it is, this microorganism has activity of decomposing the product2-keto-3-deoxy-D-gluconic acid, thus causing the yield to decrease.

As described above, no gluconate dehydratase industrially applicable inpractice has been discovered, and no industrial process for efficientlyproducing a 2-keto-3-deoxyaldonic acid has been established.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide a novelgluconate dehydratase having such a thermostability and storagestability as to be industrially used in practice and capable ofefficiently producing a 2-keto-3-deoxyaldonic acid, which is useful forpharmaceutical material, from the corresponding aldonic acid, and to aprocess for producing a 2-keto-3-deoxyaldonic acid with use of thegluconate dehydratase.

Another object of the present invention is to provide a base sequenceencoding the gluconate dehydratase, a plasmid containing the basesequence, and a cell into which a host cell is transformed with theplasmid. The base sequence is advantageously used in productions of thegluconate dehydratase and of 2-keto-3-deoxyaldonic acids using thegluconate dehydratase.

The inventors of the present invention have conducted intensive researchto find that Achromobacter xylosoxidans strain ATCC 9220 exhibits a highactivity of dehydrating gluconic acid.

A paper on the Alcaligenes sp. strain M250 said that the strain M250 isthe same type as the ATCC 9220 strain, and the gluconate dehydratasederived from Alcaligenes sp. strain M250 is reported to be unstable, asdescribed above. However, the inventors found that D-gluconic acid actedupon by Achromobacter xylosoxidans strain ATCC 9220 maintains itsactivity stably even at a reaction temperature of 50° C. and efficientlyproduces 2-keto-3-deoxy-D-gluconic acid.

Then, the inventors isolated a gluconate dehydratase from bacterialcells of this strain by various purification techniques, and allowed thegluconate dehydratase to stand at 55° C. for 2 hours. As a result, itwas found that the gluconate dehydratase is so heat-resistant as tomaintain at least 95% of the activity.

In spite of the report that the dehydration activity of the gluconatedehydratase derived from Alcaligenes sp. strain M250 is 50% degraded instorage at 0° C. for 7 days in a Tris buffer (pH 8.0 to 8.8) containing1 mM of sodium D-gluconate and 1 mM of magnesium chloride, the inventorsfound that the gluconate dehydratase of the present invention is sostable as to maintain the activity stably for one month under the sameconditions.

In addition, while it has been reported that the molecular weight of thegluconate dehydratase purified from Alcaligenes sp. strain M250determined by gel permeation chromatography is 270,000±2,500, themolecular weight of the gluconate dehydratase of the present inventionis different and 188,000±2,500.

Moreover, the inventors successfully determined the amino acid sequenceof the gluconate dehydratase shown in sequence ID No: 2 of the sequencelisting.

Furthermore, the inventors successfully produced a 2-keto-3-deoxyaldonicacid efficiently through preparing DNA containing the base sequenceshown in SEQ ID No: 1 and a cell transformed with a plasmid containing aDNA fragment having the base sequence, preparing the gluconatedehydratase so as to be active, and reacting the corresponding aldonicacid with the transformed cell or processed product from the transformedcell. Thus, the inventors have accomplished the present invention.

According to an aspect of the present invention, a gluconate dehydratasecapable of dehydrating D-gluconic acid to produce2-keto-3-deoxy-D-gluconic acid is provided. The gluconate dehydratasemaintains at least 95% of its enzyme activity after being allowed tostand in 30 mM tris(hydroxymethyl)aminomethane buffer solution with a pHof about 8.5 containing 1 mM of sodium D-gluconate and 1 mM of magnesiumchloride at 55° C. for 2 hours.

Preferably, the gluconate dehydratase is derived from Achromobacterxylosoxidans.

The gluconate dehydratase may be defined by an amino acid sequence shownin SEQ No: 2.

The gluconate dehydratase may be defined by an amino acid sequencehaving a homology of at least 70% with the amino acid sequence shown inSEQ ID No: 2.

According to another aspect of the present invention, a gene encodingthe gluconate dehydratase is provided.

The gene may be defined by a base sequence shown in SEQ ID No: 1.

According to another aspect of the present invention, a gene encodingthe gluconate dehydratase is provided which is defined by a basesequence capable of hybridizing with the foregoing gene under stringentconditions.

According to another aspect of the present invention, a plasmidcontaining any one of the genes above is provided.

The present invention is also directed to a transformed cell prepared bytransforming a host cell with the plasmid.

Preferably, the host cell is Escherichia coli.

The present invention is also directed to a process for converting analdonic acid into a corresponding 2-keto-3-deoxyaldonic acid. Theprocess includes the step of converting the aldonic acid into the2-keto-3-deoxyaldonic acid in a water-based medium with one selectedfrom the group consisting of the gluconate dehydratase, the transformedcell, and processed products from the gluconate dehydratase and thetransformed cell.

The present invention is also directed to a process for producing a2-deoxyaldonic acid. The process includes the steps of: reacting analdonic acid or a salt of the aldonic acid with one selected from thegroup consisting of the gluconate dehydratase, the transformed cell, andprocessed products from the gluconate dehydratase and the transformedcell in a water-based medium to convert the aldonic acid or the saltinto a 2-keto-3-deoxyaldonic acid; and reacting the2-keto-3-deoxyaldonic acid with an oxidizing agent in a water-basedmedium to decarboxylate and to reduce the carbon number by 1, therebyproducing the 2-deoxyaldonic acid.

The present invention is also directed to a process for producing a2-deoxyaldose. The process includes the steps of: reacting an aldonicacid or a salt of the aldonic acid with one selected from the groupconsisting of the gluconate dehydratase, the transformed cell, andprocessed products from the gluconate dehydratase and the transformedcell in a water-based medium to convert the aldonic acid or the saltinto a 2-keto-3-deoxyaldonic acid; reacting the 2-keto-3-deoxyaldonicacid with a reducing agent in a water-based medium to prepare a2-hydroxy-3-deoxyaldonic acid; and reacting the 2-hydroxy-3-deoxyaldonicacid with an oxidizing agent in a water-based medium to decarboxylateand to reduce the carbon number by 1, thereby producing the2-deoxyaldose.

The aldonic acid may be selected from the group consisting of D-gluconicacid, D-galactonic acid, D-fuconic acid, D-xylonic acid, and L-arabonicacid.

The gluconate dehydratase has a thermostability and a storage stability.The present invention provides a base sequence encoding the gluconatedehydratase, a plasmid containing the base sequence, and a celltransformed with the plasmid. By using the gluconate dehydratase or thetransformed cell, a 2-keto-3-deoxy aldonic acid and a 2-deoxyaldonicacid or 2-deoxyaldose whose carbon number is reduced by 1 can beefficiently produced.

BRIEF DESCRIPTION OF THE DRAWINGS

The FIGURE shows the structure of a plasmid containing a DNA encoding agluconate dehydratase gene.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will now be further described in detail.

A gluconate dehydratase of the present invention is heat-resistant, andacts on D-gluconic acid to catalyze a reaction for producing2-keto-3-deoxy-D-gluconic acid. More specifically, the gluconatedehydratase is so heat-resistant as to maintain at least 95% of theenzyme activity to the initial activity after being allowed to stand in30 mM Tris buffer (pH 8.5) containing 1 mM of sodium D-gluconate and 1mM of magnesium chloride at 55° C. for 2 hours.

The gluconate dehydratase of the present invention may be derived fromany type of microorganism or prepared by genetically modifying a knowngluconate dehydratase, as long as it is heat resistant and has gluconicacid dehydration activity. The gluconate dehydratase may be derived fromAchromobacter xylosoxidans strain ATCC 9220. The Achromobacterxylosoxidans strain ATCC 9220 can be provided from the American TypeCulture Collection. This strain was referred to as Alcaligenesxylosoxidans or Alcaligenes faecalis before.

The gluconate dehydratase activity in the present invention produces2-keto-3-deoxy-D-gluconic acid, using D-gluconic acid as the substrate.The activity can be determined as follows: A reaction mixture in anamount of 1 mL containing 1 mmol of a Tris buffer (pH 8.5), 40 μmol ofsodium D-gluconate, 1 μmol of magnesium chloride, and the gluconatedehydratase is allowed to react at 37° C. for 10 minutes, and 200 μL of1 M hydrochloric acid solution is added to the reaction mixture to stopthe reaction; and then, the amount of the resulting2-keto-3-deoxy-D-gluconic acid is determined by high-performance liquidchromatography (column: Shodex Asahipak NH2P-50 4E, produced by ShowaDenko, column temperature: 40° C., mobile phase: 50 mM sodium dihydrogenphosphate solution, flow rate: 1 mL/min, detection at 210 nm). An amountof enzyme for catalyzing the production of 1 μmol of2-keto-3-deoxy-D-gluconic acid for one minute is defined as 1 U. Theamount of protein may be determined by a dye-binding method using aBio-Rad protein assay kit.

The gluconate dehydratase of the present invention may have an aminoacid sequence shown in SEQ ID No: 2 of the sequence listing or an aminoacid sequence prepared by substitution in, deletion from, modificationin, insertion to, or addition to the amino acid sequence shown in SEQ IDNo: 2 of at least one amino acid, preferably several amino acids, to anextent maintaining the gluconate dehydration activity. The gluconatedehydratase of the present invention may contain a protein having ahomology of at least 70% with the amino acid sequence shown in SEQ IDNo. 2, preferably at least 80%, and more preferably at least 95%.Protein homologies can be searched with a program, such as FASTA orBLAST, in protein amino acid sequence databases, such as SWISS-PROT andPIR, and DNA databases, such as DNA Databank of JAPAN (DDBJ), EMBL, andGene-Bank, for example, on the Internet. The homology of at least 70%herein is a positive homology based on, for example, the BLAST program.

A polynucleotide encoding the gluconate dehydratase of the presentinvention contains a base sequence shown in SEQ ID No: 1. The basesequence shown in SEQ ID No: 1 in the sequence listing encodes a proteinshown in SEQ ID No: 2. The base sequence encoding the amino acidsequence shown in SEQ ID No: 2 contains not only the base sequence shownin SEQ ID No: 1, but also any base sequence based on different codons.Homologues of the polynucleotide can be prepared by appropriatesubstitution, deletion, insertion, or addition. The homologues of thepolynucleotide are prepared by substitution in, deletion from, oraddition to the base sequence shown in SEQ ID No: 1 to an extentmaintaining a predetermined enzyme activity of the gluconate dehydratasewhich is encoded by the homologues. The homologues include apolynucleotide having a base sequence capable of hybridizing understringent conditions with a polypeptide having a base sequencecomplementary to the base sequence shown in SEQ ID No: 1.

Stringent hybridization may be performed according to the methoddescribed in Molecular Cloning, Cold Spring Harbor Laboratory Press; orCurrent Protocols in Molecular Biology, Wiley Interscience. Acommercially available system, such as Gene Image System of AmershamBiosciences may also be used. Specifically, hybridization is performedas follows. A DNA or RNA molecule of a test sample transferred onto afilm is hybridized with a probe labeled according to a product protocolin a hybridization buffer designated by the protocol. The hybridizationbuffer contains 0.1 percent by weight of SDS, 5 percent by weight ofdextran sulfate, 1/20 by volume of blocking agent supplied with the kit,and 2 to 7×SSC. For the hybridization agent, a preparation having aconcentration of 5 times that of a mixture containing 100× Denhardt'ssolution, 2% (w/v) Bovine serum albumin, 2% (w/v) Ficoll™ 400, and 2%(w/v) polyvinyl pyrrolidone may be diluted to 20 times. Preferably, thehybridization is performed at a temperature in the range of 40 to 80°C., and more preferably 50 to 70° C. Then, after being incubated forseveral hours or overnight, the film is washed with a washing buffer.Washing is preferably performed at room temperature, and more preferablyat the same temperature as in hybridization. The washing buffer is asolution of 6×SSC and 0.1 percent by weight of SDS, preferably 4×SSC and0.1 percent by weight of SDS, and more preferably 1×SSC and 0.1 percentby weight of SDS. After washing with such a buffer, the DNA or RNAmolecule hybridized with the probe is identified with the label used inthe probe.

The DNA encoding the novel gluconate dehydratase of the presentinvention can be isolated by the following process, for example. GenomeDNA is purified from microorganisms. After being digested by arestriction enzyme, the DNA is fractionated according to length byultracentrifugation or electrophoresis. The fractions of the DNA arecollected and inserted into plasmids to prepare a plasmid library. Aclone exhibiting gluconic acid dehydration activity is selected from thelibrary. Thus, a plasmid containing a DNA encoding the gluconatedehydratase is obtained. By analyzing the base sequence of the plasmid,the base sequence of the DNA encoding the target gluconate dehydrataseis determined. Thus, the amino acid sequence of the gluconatedehydratase is estimated from the DNA base sequence.

The isolated DNA is inserted into an expression plasmid to prepare agluconate dehydratase expression plasmid. For example, in the case wherethe host is Escherichia coli, the DNA is inserted into pUC18, pKK223-3,pBR322, pMW119, Bluescript II SK(+), pSC101, or other expressionplasmid. Any type of organism may be used as the host fortransformation, as long as it allows the recombination vector to growstably and autonomously and characteristics of exogenous DNA to beexpressed. A typical example of the host is Escherichia coli, but is notlimited to this. Other examples of the host include bacteria, such asEscherichia, Bacillus including Bacillus subtilis, and Pseudomonas;yeasts, such as Saccharomyces, Pichia, and Candida; and mould fungi,such as Aspergillus.

In the present invention, cells transformed with the plasmid may becultured according to known information to produce the gluconatedehydratase of the present invention. For the cultivation, any culturemedium containing adequate amounts of a carbon source, a nitrogensource, and inorganic and other nutrients may be used whether asynthetic medium or a natural medium. The cultivation is performed in aculture broth containing the above-mentioned nutrients by a conventionalmethod, such as shake culture, aeration and spinner culture, continuousculture, or fed batch culture. Cultivation conditions are appropriatelyselected according to the type of culture medium and cultivation method,and are not particularly limited as long as the strain grows to producethe gluconate dehydratase.

In the process for preparing a 2-keto-3-deoxyaldonic acid, the gluconatedehydratase may be in a form of culture broth containing bacterial cellshaving gluconate dehydratase activity, of transformed cell prepared fromthe culture broth by centrifugation and collection, or of processedproduct from the transformed cell. Such processed products include anextract from or a fragmentized product of the transformed cell, productsisolated and purified from the gluconate dehydratase-active fraction ofthe extract or fragmentized product, and immobilized products in whichthe transformed cell, the extract or fragmentized product, or theisolated product is immobilized on a support.

The aldonic acid used in the present invention can be prepared by aknown process, and is also commercially available. Any aldonic acidcapable of being converted into the corresponding 2-keto-3-deoxyaldonicacid can be used. Preferred aldonic acids include D-gluconic acid,D-galactonic acid, D-fuconic acid, D-xylonic acid, and L-arabonic acid.In addition, alkali metal salts, alkaline-earth metal salts, and aminesalts of the aldonic acid may also be used.

The concentration of the aldonic acid is not particularly limited, butgenerally in the range of 1 to 500 g/L. Preferably, it is 100 g/L ormore from the viewpoint of reactivity and cost efficiency.

The temperature of the reaction for preparing the 2-keto-3-deoxyaldonicacid is set in a range in which the gluconate dehydratase can maintainthe activity, and preferably in the range of 50 to 60° C.

The pH of the reaction is set in a range in which the gluconatedehydratase can maintain the activity, and preferably in the range of 7to 9. If the pH varies during the reaction, pH can be appropriatelyadjusted.

The reaction medium may be water or a water-based medium containing abuffer solution. The buffer solution contains in water, for example, atleast one selected from the group consisting of phosphoric acid, Tris,citric acid, acetic acid, boric acid, glycine,2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid,3-morpholinopropanesulfonic acid, 2-morpholinoethanesulfonic acid,3-cyclohexylaminopropanesulfonic acid, 2-(cyclohexylamino)ethanesulfonicacid, and piperazine-N,N′-bis(2-ethanesulfonic acid) and their salts.

In order to further increase the efficiency and yield of the reaction,various types of additive may be added, if necessary. Since somegluconate dehydratases are activated by, for example, magnesium ormanganese ions, such a divalent metal may be added into the reactionliquid.

The process for producing a 2-deoxyaldonic acid includes the step ofpreparing a 2-keto-3-deoxyaldonic acid, as described above, and the stepof reacting the resulting 2-keto-3-deoxyaldonic acid with an oxidizingagent to decarboxylate into a 2-deoxyaldonic acid whose carbon number isreduced by 1.

Oxidizing agents used for the decarboxylation include hypochlorous acid,hypochlorites, and hydrogen peroxide. Preferably, hypochlorous acid or ahypochlorite is used. The hypochlorite may be sodium hypochlorite,potassium hypochlorite, calcium hypochlorite, or lithium hypochlorite,or may be prepared in a reaction system containing a metal hydroxide,such as sodium hydroxide, and chlorine.

The solvent used in the decarboxylation is not particularly limited aslong as it helps reaction proceed. Preferably, the solvent dissolves theraw materials and is, for example, water or acetic acid.

The decarboxylation is performed at a temperature between a temperaturehigher than the freezing point of the solvent and the boiling point ofthe solvent, and preferably in the range of 20 to 50° C.

The pH of the reaction liquid of the decarboxylation is in the range of4 to 6, preferably 4.5 to 6. Preferably, the pH of the reaction liquidis adjusted to such a range with an acid, simultaneously with theaddition of the hypochlorite solution or oxidizing agent. Exemplaryacids include, but not limited to, inorganic acids, such as hydrochloricacid, sulfuric acid, and phosphoric acid; lower aliphatic carboxylicacids, such as acetic acid and formic acid.

The 2-deoxyaldonic acid may be prepared by consecutive reaction in onereaction system without isolating or purifying the 2-keto-3-deoxyaldonicacid prepared in a previous step.

The process for producing a 2-deoxyaldose includes the step of preparinga 2-keto-3-deoxyaldonic acid, as described above, the step of reactingthe resulting 2-keto-3-deoxyaldonic acid with a reducing agent toprepare a 2-hydroxy-3-deoxyaldonic acid, and the step of reacting theresulting 2-hydroxy-3-deoxyaldonic acid with an oxidizing agent todecarboxylate into a 2-deoxyaldose whose carbon number is reduced by 1.

The reducing agent is not particularly limited as long as it helps thereaction proceed, and is preferably sodium borohydride. The reductionreaction may be performed by catalytic hydrogenation in the presence ofa metal catalyst, such as palladium.

The solvent used in the reduction is not particularly limited as long asit helps the reduction proceed. Preferably, the solvent dissolves theraw materials and is, for example, water.

The reduction is performed at a temperature between a temperature higherthan the freezing point of the solvent and the boiling point of thesolvent, and preferably in the range of 0 to 20° C.

The decarboxylation may be performed under the same conditions as in thedecarboxylation in the process for producing the 2-deoxyaldonic acid.

The 2-deoxyaldose may be prepared by consecutive reaction in onereaction system without isolating or purifying the reaction productsprepared previous steps.

For isolation and collection of the products 2-keto-3-deoxyaldonic acid,2-deoxyaldonic acid, and 2-deoxyaldose, a conventional method isapplied. For example, the products may be precipitated in a metal saltform or subjected to column chromatography.

EXAMPLES

The present invention will be further described with reference toexamples. However, the invention is not limited to the examples.

The amounts of the aldonic acid used in reaction and the resulting2-keto-3-deoxyaldonic acid were determined by high-performance liquidchromatography (column: Shodex Asahipak NH2P-50 4E, produced by ShowaDenko, column temperature: 40° C., mobile phase: 50 mM sodium dihydrogenphosphate solution, flow rate: 1 mL/min, detection at 210 nm).

Example 1

Cultivation of Achromobacter xylosoxidans Strain ATCC 9220:

The bacterial cells of Achromobacter xylosoxidans strain ATCC 9220 grownin a broth culture medium in advance were inoculated into a liquidculture medium (pH 7.0) containing 10 g/L of sodium D-gluconate, 5 g/Lof yeast extract, 5 g/L of polypeptone, 3 g/L of sodium chloride, and0.2 g/L of magnesium sulfate heptahydrate, and subjected to aeration andspinner culture at 30° C. for 20 hours. The cultured cells werecollected by centrifugation to yield bacterial cells having gluconatedehydratase activity.

Example 2

Synthesis of 2-keto-3-deoxy-D-gluconic Acid with Achromobacterxylosoxidans Strain ATCC 9220:

In 200 mL of 50 mM Tris buffer (pH 7.0, containing 1 mM of sodiumD-gluconate and 1 mM of magnesium chloride), 120 g of the wet bacterialcell of the Achromobacter xylosoxidans prepared in Example 1 weredispersed, and crushed with an ultrasonic cell crusher to prepare acrude enzyme liquid. The crude enzyme liquid was added to a solution of1 mmol of sodium magnesium in 600 mL of water. After being adjusted topH 8.5 with 6 M sodium hydroxide solution, the mixture was allowed toreact at 50° C. During the reaction, the pH of the reaction mixture wasadjusted to 8.5 by appropriately adding 2 M sodium hydroxide solution.After the reaction for 40 hours, there was not D-gluconic acid but 95 gof 2-keto-3-deoxy-D-gluconic acid in the reaction mixture.

After the completion of reaction, solid contents derived from thebacterial cells were removed from the reaction mixture bycentrifugation, and the supernatant liquor was filtrated through anultrafilter membrane (Biomax-10, produced by Millipore). The filtratewas passed through an ion-exchange column using Dowex 1×8 (200-400meshes, OH form, produced by Dow Chemical) and eluted with 50 mLhydrochloric acid solution. The collected eluate was concentrated underreduced pressure, and then neutralized with a potassium hydroxidesolution to yield 346.7 g of an aqueous solution containing 73.8 g ofpotassium 2-keto-3-deoxy-D-gluconate.

Example 3

Purification of Gluconate Dehydratase Derived from Achromobacterxylosoxidans Strain ATCC 9220 and Determination of N-terminus Amino AcidSequence:

The bacterial cells obtained in Example 1 were suspended in 50 mM Trisbuffer containing 1 mM of sodium D-gluconate and 1 mM of magnesiumchloride (pH 7.0, referred to as the buffer in Example 3), and crushedwith an ultrasonic cell crusher. The suspension was subjected torefrigerated centrifugation and the supernatant fluid of the suspensionwas collected to obtain a cell-free extract. Into the cell-free extractwas added 1% of streptomycin sulfate, and the mixture was stirred for 30minutes to form a precipitate. After removing the precipitate bycentrifugation, ammonium sulfate was added to the supernatant and 20 to60% saturated fraction was collected. The ammonium sulfate fraction wasdemineralized and concentrated through an ultrafilter membrane(Ultrafree-15 with a 100,000-molecular weight cut-off, produced byMillipore), then passed through a column DEAE-Sepharose FF (produced byAmersham Biosciences), and eluted by a linear concentration gradientbetween the buffer and another buffer containing 1 M of NaCl. Theresulting active fraction was collected, and into which ammonium sulfatewas added at a concentration of 1 M. The mixture was passed through acolumn Phenyl-Sepharose HP (produced by Amersham Biosciences) and thuseluted by a linear concentration gradient between the buffer and anotherbuffer containing 1 M of ammonium sulfate. The resulting active fractionwas collected and passed through a column Superose 12HR (produced byAmersham Biosciences), and was thus eluted with a Tris buffer containing0.15 M of NaCl. The active fraction was collected, and into whichammonium sulfate was added at a concentration of 0.5 M. The mixture waspassed through the column Phenyl-Sepharose HP, and thus eluted by alinear concentration gradient between the buffer and another buffercontaining 0.5 M of ammonium sulfate. The active fraction was collected,and into which ammonium sulfate was added at a concentration of 0.5 M.The mixture was passed through the column Phenyl-Sepharose HP, and thuseluted by a linear concentration gradient between the buffer and anotherbuffer containing 0.5 M of ammonium sulfate. The active fraction wascollected and subjected to sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. As a result, a single band was identified at about 60kDa. The active fraction was dialyzed against 30 mM Tris buffer (pH 8.5)containing 1 mM of sodium D-gluconate and 1 mM of magnesium chloride toexchange the buffer. The resulting enzyme solution was used as apurified gluconate dehydratase solution. Table 1 show the purificationsummary.

The N-terminus amino acid sequence of the protein at about 60 kDa wasanalyzed, and determined to beThr-Asp-Thr-Pro-Arg-Lys-Leu-Arg-Ser-Gln-Lys-Trp-Phe-Asp-Asp, as shown inSEQ ID No. 3 of the sequence listing.

TABLE 1 Purification of Gluconate Dehydratase Total Total Specific Puri-protein activity activity fication mg unit unit/mg degree Yield %Cell-free extract 1322 284.8 0.22 1 100 Ammonium sulfate 814.1 160.20.20 0.9 56.3 fractionation (20-60% sat.) DEAE Sepharose FF 165.5 43.10.26 1.2 15.1 1st Phenyl Sepharose HP 6.9 17.1 2.49 11.3 6.0 Superose 12HR 2.9 20.9 7.23 32.9 7.3 2nd Phenyl 1.3 20.3 15.5 70.5 7.1 Sepharose HP3rd Phenyl Sepharose HP 1.3 17.0 13.6 63.3 6.0

Example 4

Thermostability of Gluconate Dehydratase Derived from Achromobacterxylosoxidans Strain ATCC 9220:

Samples of the purified gluconate dehydratase solution prepared inExample 3, which was dissolved in the 30 mM Tris buffer (pH 8.5)containing 1 mM sodium D-gluconate and 1 mM magnesium chloride, wereallowed to stand for 30 minutes, 2 hours, or 12 hours at 4, 20, 30, 40,50, 55, 60, 65, or 70° C. Another sample was allowed to stand at 4° C.for 7 days and 30 days. Then, each sample subjected to heat treatment asabove was reacted with 1 mL of a liquid containing 1 mmol of Tris buffer(pH 8.5), 40 μmol of sodium D-gluconate, and 1 μmol of magnesiumchloride at 37° C. After 10 minutes, 200 μL of 1 M hydrochloric acidsolution was added to the reaction mixture to stop the reaction. Then,the amount of 2-keto-3-deoxy-D-gluconic acid produced was determined byhigh-performance liquid chromatography, and the reaction rate wascalculated. The results are shown in Table 2 by relative activities tothe enzyme activity before heat treatment which is represented as 100.The activity of 100% was maintained after treatment at 55° C. for 2hours, and it is also stably maintained for 30 days at 4° C.

TABLE 2 Thermostability of Gluconate Dehydratase Time 30 min 2 h 12 h 7day 30 day Temperature Remaining activity (%)  4° C. 100 100 100  100105 20° C. 103 — — — — 30° C. 103 — 90 — — 35° C. 107 — 91 — — 40° C.105 — 90 — — 45° C. 109 — — — — 50° C. 103 — 77 — — 55° C. 103 104 — — —60° C. 85  51 — — — 65° C. 75 — — — — 70° C. 41 — — — —

Example 5

Determination of Internal Amino Acid Sequence of Gluconate DehydrataseDerived from Achromobacter xylosoxidans Strain ATCC 9220:

The purified gluconate dehydratase solution (124 μg in terms of protein)prepared in Example 3 was freeze-dried, then dissolved in 100 μL of 0.5M Tris buffer (pH 8.4) containing 6 M of guanidine hydrochloride, andwarmed at 37° C. for 15 minutes. To the solution was added 10 μL of 0.5M Tris buffer (pH 8.4) containing 0.2 M of dithiothreitol and 6 M ofguanidine hydrochloride. The solution was then warmed at 60° C. for 1hour. To the solution was added 10 μL of 0.5 M Tris buffer (pH 8.4)containing 0.4 M of iodoacetic acid and 6 M of guanidine hydrochloride.The solution was then warmed at 37° C. for 15 minutes. The resultingsolution was passed through a Quick Spin column (produced by RocheDiagnostics) filled with Sephadex G-50 equilibrated with 0.5 M ammoniumhydrogen carbonate solution (pH 7.8) previously containing 8 M of ureato demineralize, and thus 400 μL of solution was obtained. To thissolution was added 1 μL of 2 mg/mL V8 protease (produced by Wako PureChemical Industries) to allowed to react at 30° C. for 23 hours. Thereaction mixture was subjected to reversed-phase high performance liquidchromatography using a column Vydac 214TP54 PROTEIN C4 (produced byAgilent) to isolate the product peptide by a concentration gradientbetween 0.1% trifluoroacetic acid solution and 90% acetonitrile solutioncontaining 0.1% of trifluoroacetic acid, and a fraction of the peptidewas sampled. The N-terminus amino acid sequence of the peptide wasanalyzed, and determined to beAla-Arg-Ala-Ile-Val-Phe-Glu-Gly-Pro-Glu-Asp-Tyr-His-Ala-Arg, as shown inSEQ ID No: 4 of the sequence listing.

Example 6

DNA Encoding Gluconate Dehydratase from Achromobacter xylosoxidansStrain ATCC 9220:

Genome DNA was prepared from the bacterial cells of Achromobacterxylosoxidans strain ATCC 9220 prepared in Example 1 according to themethod for isolation of bacterial genome DNA described in “Kiso-KagakuJikken-Hou 2, Chushutsu, Bunri, Seisei (Basic Chemical Experiments 2,Extraction, Separation, and Purification)”, Kouichi Anan, et al.,published by Maruzen Company. Polymerase chain reaction (PCR) wasperformed to prepare DNA fragment of about 1.2 kb, using the genome DNAas a temperate, and an oligonucleotide defined by SEQ ID No: 5synthesized according to the N-terminus amino acid sequence determinedin Example 3 and an oligonucleotide defined by SEQ ID No: 6 synthesizedaccording to the internal amino acid sequence determined in Example 5 asprimer. The genome DNA was digested with typical restriction enzymes,and totally southern-hybridized with a probe prepared by labeling theDNA fragment of about 1.2 kb. As a result, when the genome DNA wascompletely digested with a restriction enzyme Pst I, a positive signalwas found in a fragment of about 4 kb. The DNA fragments obtained bycompletely digesting the genome DNA with the restriction enzyme Pst Iwere subjected to concentration gradient ultracentrifugation tofractionate according to length, and a fraction mainly containing 4-kbDNA fragments was collected. The fraction was subjected to DNA ligationwith a vector pUC118 whose 5′-terminus was dephosphorylated by digestingwith the restriction enzyme Pst I to prepare a plasmid library. Atransformant into which Escherichia coli DH5α was transformed with theplasmid library was applied onto a LB (Luria-Bertani) agarose mediumcontaining 50 μg/mL of Ampicillin and static-cultured to producecolonies. Colony hybridization was performed with the labeled probe of1.2-kb DNA fragment, and a colony exhibiting a positive signal wasisolated. A plasmid was collected from the positive colony, and the basesequence of the resulting plasmid was analyzed. For the analysis of thebase sequence, BigDye Teminator Cycle Sequencing kit and GeneticAnalyzer 310, produced by Applied Biosystems, were used. According tobase sequence information, two types of primer, oligonucleotides shownin SEQ ID Nos: 7 and 8, were designed. The primer of SEQ ID No: 7 wasprovided with a restriction enzyme HindIII site; the primer of SEQ IDNo: 8, restriction enzyme XbaI site. PCR was performed to expand theregion containing the DNA encoding the gluconate dehydratase, using thegenome DNA as the template. The PCR product was subjected to agarose gelelectrophoresis and a band having a targeted mobility was cut out. Thus,the DNA was extracted from the gel with Qiaqick produced by Qiagen. Theextract was subjected to DNA ligation with a vector pMW119 whose5′-terminus was dephosphorylated by digesting with the restrictionenzymes HindIII and XbaI to yield a plasmid containing the DNA encodingthe gluconate dehydratase.

Example 7

Determination of DNA Encoding Gluconate Dehydratase Gene:

According to an examination, it was found that plasmid prepared inExample 6 has a physical map as shown in the FIGURE. Also, the basesequence of the plasmid was analyzed. For the determination of the basesequence, BigDye Teminator Cycle Sequencing kit and Genetic Analyzer310, produced by Applied Biosystems, were used. As a result, the entirebase sequence of the DNA encoding the gluconate dehydratase gene, shownin SEQ ID No: 1 was obtained. The base sequence of the gluconatedehydratase gene was translated into an amino acid sequence, and theresult is shown in SEQ ID No: 2. The amino acid sequence of itsN-terminus was in agreement with that of the N-terminus described inExample 3.

Example 8

Synthesis of 2-keto-3-deoxy-D-gluconic Acid with Escherichia coliTransformed with DNA Containing the Gene of Gluconate DehydrataseDerived from Achromobacter xylosoxidans Strain ATCC 9220:

Escherichia coli K-12 W3110 transformed with the plasmid of Example 7,shown in the FIGURE, was shake-cultured in a LB liquid medium containing50 μg/mL of Ampicillin at 37° C. overnight. After the cultivation,bacterial cells were collected by centrifugation. Then, 50.0 g ofreaction mixture was allowed to react at 50° C. which contained 0.3 g ofthe resulting wet bacterial cells, 6.0 g of sodium D-gluconate, 50 μmolof magnesium chloride, and 2.5 mmol of Tris buffer (pH 8.5). After thereaction for 24 hours, there was not D-gluconic acid but 4.8 g of2-keto-3-deoxy-D-gluconic acid in the reaction mixture.

Example 9

Synthesis of 2-keto-3-deoxy-D-galactonic Acid with Escherichia coliTransformed with DNA Containing the Gene of Gluconate DehydrataseDerived from Achromobacter xylosoxidans Strain ATCC 9220:

Escherichia coli K-12 W3110 transformed with the plasmid of Example 7,shown in the FIGURE, was shake-cultured in a LB liquid medium containing50 μg/mL of Ampicillin at 37° C. overnight. After the cultivation,bacterial cells were collected by centrifugation. Then, 50.0 g ofreaction mixture was allowed to react at 50° C. which contained 0.6 g ofthe resulting wet bacterial cells, 6.0 g of sodium D-galactonate, 50μmol of magnesium chloride, and 2.5 mmol of Tris buffer (pH 8.5). Afterthe reaction for 24 hours, there was not D-galactonic acid but 4.5 g of2-keto-3-deoxy-D-galactonic in the reaction mixture.

Example 10

Synthesis of 2-keto-3-deoxy-D-xylonic Acid with Escherichia coliTransformed with DNA Containing the Gene of Gluconate DehydrataseDerived from Achromobacter xylosoxidans Strain ATCC 9220:

Escherichia coli K-12 W3110 transformed with the plasmid of Example 7,shown in the FIGURE, was shake-cultured in a LB liquid medium containing50 μg/mL of Ampicillin at 37° C. overnight. After the cultivation,bacterial cells were collected by centrifugation. Then, 110.0 g ofreaction mixture was allowed to react at 50° C. which contained 0.7 g ofthe resulting wet bacterial cells, 13.0 g of ammonium D-xylonate, 50μmol of magnesium chloride, and 5.5 mmol of Tris buffer (pH 8.5). Afterthe reaction for 24 hours, there was not D-xylonic acid but 9.9 g of2-keto-3-deoxy-D-xylonic acid in the reaction mixture.

Example 11

Synthesis of 2-keto-3-deoxy-L-arabonic Acid with Escherichia coliTransformed with DNA Containing the Gene of Gluconate DehydrataseDerived from Achromobacter xylosoxidans Strain ATCC 9220:

Escherichia coli K-12 W3110 transformed with the plasmid of Example 7,shown in the FIGURE, was shake-cultured in a LB liquid medium containing50 μg/mL of Ampicillin at 37° C. overnight. After the cultivation,bacterial cells were collected by centrifugation. Then, 10.0 g ofreaction mixture was allowed to react at 50° C. which contained 0.7 g ofthe resulting wet bacterial cells, 1.0 g of sodium L-arabonate, 10 μmolof magnesium chloride, and 500 μmol of Tris buffer (pH 8.5). After thereaction for 24 hours, there was not L-arabonic acid but 0.7 g of2-keto-3-deoxy-L-arabonic acid in the reaction mixture.

Example 12

Synthesis of 2-deoxyribose with Sodium Gluconate as Aldonate:

Escherichia coli K-12 W3110 transformed with the plasmid of Example 7,shown in the FIGURE, was shake-cultured in a LB liquid medium containing50 μg/mL of Ampicillin at 37° C. overnight. After the cultivation,bacterial cells were collected by centrifugation. Then, 3.3 g of theresulting wet bacterial cell was added to 400 g of reaction mixturewhich contained 130 g of sodium D-gluconate and 10 μmol of magnesiumchloride and whose pH was adjusted to 8.5 with sodium hydroxide, andallowed to react at 40° C. After the reaction for 24 hours, there wasnot D-gluconic acid but 106 g of 2-keto-3-deoxy-D-gluconic acid in thereaction mixture.

The resulting reaction mixture, which contained2-keto-3-deoxy-D-gluconic acid, was cooled to 10° C. while beingstirred, and 6.0 g of sodium borohydride was slowly added to the mixturewith care to avoid bubbling. Then, the reaction was continued at 10° C.for 2 hours. Consequently, 100 g of 2-hydroxy-3-deoxy-D gluconic acidwas produced in the reaction mixture.

The pH of the resulting reaction mixture containing the2-hydroxy-3-deoxy-D-gluconic acid was adjusted to 5.0 with 35%hydrochloric acid solution. Into the reaction mixture was dripped 377 gof 13% sodium hypochlorite solution to perform decarboxylation over aperiod of 1 hour with the reaction temperature adjusted to 35° C. The pHof the reaction mixture at this time was adjusted between 5 and 6 withacetic acid. After adding the sodium hypochlorite solution, the reactionwas carried out for 1 hour to yield 69 g of 2-deoxy-D-ribose. The yieldfrom sodium D-gluconate was 86.3%.

Example 13

Synthesis of Mixture of 2-deoxy-D-ribonolactone and Sodium2-deoxy-D-ribonate using Sodium Gluconate as Aldonate:

Escherichia coli K-12 W3110 transformed with the plasmid of Example 7,shown in the FIGURE, was shake-cultured in a LB liquid medium containing50 μg/mL of Ampicillin at 37° C. overnight. After the cultivation,bacterial cells were collected by centrifugation. Then, 3.3 g of theresulting wet bacterial cell was added to 530 g of reaction mixturewhich contained 130 g of sodium D-gluconate and 10 μmol of magnesiumchloride and whose pH was adjusted to 8.5 with sodium hydroxide, andallowed to react at 40° C. After the reaction for 24 hours, there wasnot D-gluconic acid but 106 g of 2-keto-3-deoxy-D-gluconic acid in thereaction mixture.

The pH of the 2-keto-3-deoxy-D-gluconic acid solution was adjusted to9.0 with 30% sodium hydroxide, and then to 5.0 with concentratedhydrochloric acid. While the pH was adjusted between 4.5 and 5.0 withconcentrated hydrochloric acid under water-cooling, 473 g of sodiumhypochlorite solution (12.2 percent by weight) was dripped over a periodof 1 hour. After the completion of reaction, sodium hydrogencarbonatewas added to adjust the pH to 8.0. The resulting reaction mixture wasconcentrated under reduced pressure at 50° C. Methanol was added to theresidue and the resulting inorganic salt was removed by filtration. Thisstep was repeated two times. The solvent of the filtrate was removed byconcentration to yield 106 g of a mixture of 2-deoxy-D-ribonolactone andsodium 2-deoxy-D-ribonate. In 27 g of water was dissolved 2.7 g of theresulting mixture. After being demineralized through a cation exchangeresin (Amberlite IR-120 plus), the solution was adjusted to pH 0.9 with12 N HCl and stirred at room temperature for 12 hours. The resultingreaction solution was concentrated under reduced pressure, and thenpurified by silica gel column chromatography (eluant composition:chloroform/methanol=10/1 to 5/1)to yield 2.0 g of2-deoxy-D-ribonolactone syrup quantitatively.

Example 14

Synthesis of Sodium 3,4-dihydroxybutanate with Sodium Xylonate asAldonate:

Escherichia coli K-12 W3110 transformed with the plasmid of Example 7,shown in the FIGURE, was shake-cultured in a LB liquid medium containing50 μg/mL of Ampicillin at 37° C. overnight. After the cultivation,bacterial cells were collected by centrifugation. Then, 0.5 g of theresulting wet bacterial cell was added to 53 g of reaction mixture whichcontained 13 g of sodium D-xylonate and 10 μmol of magnesium chlorideand whose pH was adjusted to 8.5 with sodium hydroxide, and allowed toreact at 40° C. After the reaction for 24 hours, there was not D-xylonicacid but 9.8 g of 2-keto-3-deoxy-D-xylonic acid in the reaction mixture.

The pH of the 2-keto-3-deoxy-D-xylonic acid solution was adjusted to 5.0with concentrated hydrochloric acid. While the pH was adjusted between4.5 and 5.0 with concentrated hydrochloric acid under water-cooling, 53g of sodium hypochlorite solution (12.2 percent by weight) was drippedover a period of 1 hour. After the completion of reaction, sodiumhydrogencarbonate was added to adjust the pH to 8.0. The resultingreaction mixture was concentrated under reduced pressure at 50° C.Methanol was added to the residue and the resulting inorganic salt wasremoved by filtration. This step was repeated two times. The solvent ofthe filtrate was removed by concentration to yield 9.4 g of sodium3,4-dihydroxybutanate.

1. A process for producing a 2-deoxyaldonic acid, comprising the stepsof: reacting in a water based medium an aldonic acid or salt thereofwith a gluconate dehydratase capable of dehydrating D-gluconic acid toproduce 2-keto-3-deoxy-D-gluconic acid, thereby converting said aldonicacid or salt thereof into a 2-keto-3-deoxyaldonic acid; and reacting ina water based medium said 2-keto-3-deoxyaldonic acid with an oxidizingagent, thereby decarboxylating said 2-keto-3-deoxyaldonic acid andreducing the carbon number of said 2-keto-3-deoxyaldonic acid by 1 toproduce said 2-deoxyaldonic acid; wherein: said gluconate dehydratasemaintains at least 95% of the enzyme activity thereof after beingallowed to stand in 30 mM tris(hydroxymethyl) aminomethane buffersolution with a pH of about 8.5 containing 1 mM of sodium D-gluconateand 1 mM of magnesium chloride at 55° C. for 2 hours; and said gluconatedehydratase comprises the amino acid sequence shown in SEQ ID NO:2.
 2. Aprocess for producing a 2-deoxyaldose, comprising the steps of: reactingin a water-based medium an aldonic acid or salt thereof with a gluconatedehydratase capable of dehydrating D-gluconic acid to produce2-keto-3-deoxy-D-gluconic acid, thereby converting said aldonic acid orsalt thereof into a 2-keto-3-deoxyaldonic acid; reacting in awater-based medium said 2-keto-3-deoxyaldonic acid with a reducing agentto produce a 2-hydroxy-3-deoxyaldonic acid; and reacting in awater-based medium said 2-hydroxy-3-deoxyaldonic acid with an oxidizingagent, thereby decarboxylating said 2-hydroxy-3-deoxyaldonic acid andreducing the carbon number of said 2-hydroxy-3-deoxyaldonic acid by 1 toproduce said 2-deoxyaldose; wherein: said gluconate dehydratasemaintains at least 95% of the enzyme activity thereof after beingallowed to stand in 30 mM tris(hydroxymethyl) aminomethane buffersolution with a pH of about 8.5 containing 1 mM of sodium D-gluconateand 1 mM of magnesium chloride at 55° C. for 2 hours; and said gluconatedehydratase comprises the amino acid sequence shown in SEQ ID NO:2.
 3. Aprocess for converting an aldonic acid into a corresponding2-keto-3-deoxyaldonic acid, comprising the step of: reacting in a waterbased medium an aldonic acid or salt thereof with (a) a transformed cellprepared by transforming a host cell with a plasmid containing a geneencoding a gluconate dehydratase capable of dehydrating D-gluconic acidto produce 2-keto-3-deoxy-D-gluconic acid, or (b) processed productsfrom the transformed cell; wherein: said gluconate dehydratase maintainsat least 95% of the enzyme activity thereof after being allowed to standin 30 mM tris(hydroxymethyl) aminomethane buffer solution with a pH ofabout 8.5 containing 1 mM of sodium D-gluconate and 1 mM of magnesiumchloride at 55° C. for 2 hours; and said gluconate dehydratase comprisesthe amino acid sequence shown in SEQ ID NO:2.
 4. The process accordingto claim 3, wherein the aldonic acid is selected from the groupconsisting of D-gluconic acid, D-galactonic acid, D-fuconic acid,D-xylonic acid, and L-arabonic acid.
 5. A process for producing a2-deoxyaldonic acid, comprising the steps of: reacting in a water basedmedium an aldonic acid or salt thereof with (a) a transformed cellprepared by transforming a host cell with a plasmid containing a geneencoding a gluconate dehydratase capable of dehydrating D-gluconic acidto produce 2-keto-3-deoxy-D-gluconic acid, or (b) processed productsfrom the transformed cell, thereby converting said aldonic acid or saltthereof into a 2-keto-3-deoxyaldonic acid; and reacting in a water basedmedium said 2-keto-3-deoxyaldonic acid with an oxidizing agent, therebydecarboxylating said 2-keto-3-deoxyaldonic acid and reducing the carbonnumber of said 2-keto-3-deoxyaldonic acid by 1 to produce said2-deoxyaldonic acid; wherein: said gluconate dehydratase maintains atleast 95% of the enzyme activity thereof after being allowed to stand in30 mM tris(hydroxymethyl) aminomethane buffer solution with a pH ofabout 8.5 containing 1 mM of sodium D-gluconate and 1 mM of magnesiumchloride at 55° C. for 2 hours; and said gluconate dehydratase comprisesthe amino acid sequence shown in SEQ ID NO:2.
 6. The process accordingto claim 5, wherein the aldonic acid is selected from the groupconsisting of D-gluconic acid, D-galactonic acid, D-fuconic acid,D-xylonic acid, and L-arabonic acid.
 7. A process for producing a2-deoxyaldose, comprising the steps of: reacting in a water based mediuman aldonic acid or salt thereof with (a) a transformed cell prepared bytransforming a host cell with a plasmid containing a gene encoding agluconate dehydratase capable of dehydrating D-gluconic acid to produce2-keto-3-deoxy-D-gluconic acid, or (b) processed products from thetransformed cell, thereby converting said aldonic acid or salt thereofinto a 2-keto-3-deoxyaldonic acid; reacting in a water based medium said2-keto-3-deoxyaldonic acid with a reducing agent to produce a2-hydroxy-3-deoxyaldonic acid; and reacting in a water based medium said2-hydroxy-3-deoxyaldonic acid with an oxidizing agent, thereby producingthereby decarboxylating said 2-hydroxy-3-deoxyaldonic acid and reducingthe carbon number of said 2-hydroxy-3-deoxyaldonic acid by 1 to producesaid 2-deoxyaldose; wherein: said gluconate dehydratase maintains atleast 95% of the enzyme activity thereof after being allowed to stand in30 mM tris(hydroxymethyl) aminomethane buffer solution with a pH ofabout 8.5 containing 1 mM of sodium D-gluconate and 1 mM of magnesiumchloride at 55° C. for 2 hours; and said gluconate dehydratase comprisesthe amino acid sequence shown in SEQ ID NO:2.
 8. The process accordingto claim 7, wherein the aldonic acid is selected from the groupconsisting of D-gluconic acid, D-galactonic acid, D-fuconic acid,D-xylonic acid, and L-arabonic acid.
 9. The process according to claim1, wherein the aldonic acid is selected from the group consisting ofD-gluconic acid, D-galactonic acid, D-fuconic acid, D-xylonic acid, andL-arabonic acid.
 10. The process according to claim 2, wherein thealdonic acid is selected from the group consisting of D-gluconic acid,D-galactonic acid, D-fuconic acid, D-xylonic acid, and L-arabonic acid.11. A process for converting an aldonic acid into a corresponding2-keto-3-deoxyaldonic acid, comprising the step of: reacting in a waterbased medium an aldonic acid or salt thereof with a gluconatedehydratase capable of dehydrating D-gluconic acid to produce2-keto-3-deoxy-D-gluconic acid; wherein: said gluconate dehydratasemaintains at least 95% of the enzyme activity thereof after beingallowed to stand in 30 mM tris(hydroxymethyl) aminomethane buffersolution with a pH of about 8.5 containing 1 mM of sodium D-gluconateand 1 mM of magnesium chloride at 55° C. for 2 hours; and said gluconatedehydratase comprises the amino acid sequence shown in SEQ ID NO:2. 12.The process according to claim 11, wherein the aldonic acid is selectedfrom the group consisting of D-gluconic acid, D-galactonic acid,D-fuconic acid, D-xylonic acid, and L-arabonic acid.